SPIE Proceedings [SPIE SPIE Optical Metrology 2013 - Munich, Germany (Monday 13 May 2013)] Optics for Arts, Architecture, and Archaeology IV - Multiphoton microscopy: an efficient

Multiphoton microscopy, an efficient tool for in situ study of cultural heritage artifacts

Gaël Latoura,*, Jean-Philippe Echardb, Marie Didierb, Marie-Claire Schanne-Kleina

a Laboratory for Optics and Biosciences, Ecole Polytechnique, CNRS, INSERM U696, 91128, Palaiseau, France

b Laboratoire de recherche et de restauration, Musée de la musique, Cité de la musique, 221 avenue Jean-Jaurès, 75019, Paris, France

ABSTRACT

We present multimodal nonlinear optical imaging of historical artifacts by combining Two-Photon Excited Fluorescence (2PEF) and Second Harmonic Generation (SHG) microscopies. Three-dimensional (3D) non-contact laser-scanning imaging with micrometer resolution is performed without any preparation of the objects under study. 2PEF signals are emitted by a wide range of fluorophores such as pigments and binder, which can be discriminated thanks to their different emission spectral bands by using suitable spectral filters in the detection channel. SHG signals are specific for dense non-centrosymmetric organizations such as the crystalline cellulose within the wood cell walls. We also show that plaster particles exhibit SHG signals. These particles are bassanite crystals with a non-centrosymmetric crystalline structure, while the other types of calcium sulphates exhibit a centrosymmetric crystalline structure with no SHG signal.

In our study, we first characterize model single-layered samples: wood, gelatin-based films containing plaster or cochineal lake and sandarac film containing cochineal lake. We then study multilayered coating systems on wood and show that multimodal nonlinear microscopy successfully reveals the 3D distribution of all components within the stratified sample. We also show that the fine structure of the wood can be assessed, even through a thick multilayered varnish coating. Finally, in situ multimodal nonlinear imaging is demonstrated in a historical violin.

SHG/2PEF imaging thus appears as an efficient non-destructive and contactless 3D imaging technique for in situ investigation of historical coatings and more generally for wood characterization and coating analysis at micrometer scale.

Keywords: multiphoton microcopy, 3D imaging, fluorescence, second harmonic generation, varnish, wood, music instrument

1. INTRODUCTION Multiphoton microscopy, also called non-linear optical microscopy, is a widely used imaging technique for biomedical studies. This technique performs three-dimensional (3D) contactless imaging with micrometer-scale resolution based on an intrinsic optical sectioning, without any preparation of the sample. These properties are also very attractive for the study of cultural heritage artifacts, a field where non invasive and non destructive methods are greatly favored. Many artifacts of our cultural heritage are coated with transparent (varnishes) or semi-transparent (glazes, paint layers) coatings, which stratigraphy and composition are very difficult to characterize in situ.

Since few years, Optical Coherence Tomography (OCT) is a well-established technique as contactless 3D imaging tool [1, 2]. Nevertheless, the discrimination of the various components is strongly limited with OCT because the contrast of the recorded images is based on reflectance signals. Some attempts have been performed to obtain spectral information from the raw OCT data on artists’ materials [3, 4]. Nevertheless, OCT is characterized by an intrinsic tradeoff between the spatial resolution and the spectral resolution, strongly limiting spectral discrimination of the different materials.

A key advantage of multiphoton microscopy is its multimodal capability with different modes of contrast that are directly linked to the chemical nature of the materials under study. Few studies have been performed to explore the potential of this optical technique in the field of the investigation of cultural heritage artifacts [5]. We then present two- * [email protected], www.lob.polytechnique.fr

Invited Paper

Optics for Arts, Architecture, and Archaeology IV, edited by Luca Pezzati,Piotr Targowski, Proc. of SPIE Vol. 8790, 87900L · © 2013 SPIE

CCC code: 0277-786X/13/$18 · doi: 10.1117/12.2020221

Proc. of SPIE Vol. 8790 87900L-1

Downloaded From: http://proceedings.spiedigitallibrary.org/ on 09/13/2013 Terms of Use: http://spiedl.org/terms

rWi

photon excited fluorescence (2PEF) images and second harmonic generation (SHG) images from a short selection of widely used artists’ materials in order to demonstrate the interest of this technique. Model samples have been prepared with single and stratified layers. Finally, in situ investigation of a historical violin is shown.

2. MATERIALS AND METHOD 2.1 Multiphoton microscope setup

Multiphoton microscopy imaging was performed using a custom-built laser scanning upright microscope [6, 7] (see Figure 1.a). Titanium:sapphire femtosecond laser was tuned at 860 nm for the excitation. Rather than water or oil immersion objectives widely used in biomedical imaging, an air objective was used for non-contact imaging (20x, NA 0.95, Olympus). Well-suited filters have been selected for SHG detection at exactly half the excitation wavelength (427/10 interferential filters, Semrock). 2PEF signals were detected in two different spectral bands: around 485 nm (485/70 bandpass filter, Semrock) and above 590 nm (RG590 high-pass filter, Schott) [7]. All the images presented in this article are in false colors: 2PEF signals in red and SHG signals in green. Laser power at the objective focus was 8 to 20 mW, without any observable damage in the various studied samples. The spatial resolution reached with our microscope is approximately 0.45 µm laterally and 1.6 µm axially near the sample surface.

a b

2PEF

SHG

Filters

Objective(20x, NA 0.75)

Condenser

Filters

Sample

SHG

Excitation beam @860 nm

YX

XY scanning

backward detection

forward detection removable

z

Figure 1. Experimental setup. (a) Multiphoton microscope with backward detection channels (2PEF and SHG) and a forward removable SHG detection channel. Air objective was used for non-contact imaging. (b) Picture of the microscope for in situ investigation of a violin.

2.2 Samples

Studied materials have been selected as widely used artists’ materials. Maple wood (Acer pseudoplatanus) is studied as shavings and then as bulky samples. Two types of film-making materials were used: gelatin-based glue (GT58, Laverdure, Paris) and sandarac resin from restoration materials (Musée de la musique laboratory). Cochineal lake pigments (prepared at the Musée de la musique laboratory according to a historical recipe) and plaster fillers (plaster of Paris, Staturoc, Rougier & Plé, Paris) were incorporated in these binders. Single layers and stratified layers composed of the previous materials were prepared.

Finally, in situ analysis of a violin (Neuner & Hornsteiner, Mittenwald, Germany, early 20th century, private collection) was performed. For this experiment the forward detection module was removed to let enough place for a violin as shown in Figure 1.b.



3. MULTIPHOTON IMAGING OF CULTURAL HERITAGE ARTIFACTS 3.1 Wood

A wood shaving was placed between two thin glass coverslips. Both forward and backward SHG signals were detected simultaneously to 2PEF signals (high-pass GG5 filter). Strong 2PEF signals from lignin within the wood cell walls were detected (Figure 2.a). Strong SHG signals from cellulose were also detected with similar signal intensities in both detection directions (see Figure 2.b and 2.c for respectively forward and backward SHG signals). These images show that combined SHG/2PEF microscopies reveal the composition of the wood cell walls with a micrometer-scale resolution, in agreement with previous reports [8]. Moreover, cellulose imaging could be further improved by using polarization-resolved SHG to quantify the orientation and the organization of the crystalline cellulose at a submicrometer-scale resolution as already performed in collagen fibrils [6, 9].

2PEF (GG5 filter) Forward-SHG Backward-SHGa b c

Figure 2. Multiphoton imaging of maple wood. (a) 2PEF signals from lignin. (b) Forward and (c) backward SHG signals from cellulose. Scale bar: 100 µm.

3.2 Monolayers: plaster, cochineal lake, sandarac and gelatin

The first monolayer under study was a sandarac film containing cochineal lake pigments. The two distinct 2PEF spectral detection channels allowed the discrimination of these materials. 2PEF signals from cochineal lake pigments were detected around 485 nm, whereas strong 2PEF signals of sandarac film were obtained above 590 nm (see first row in Figure 3). These 2PEF emission spectral properties are consistent with published fluorescence properties of these materials [10, 11].

Secondly, we studied a gelatin-based film containing plaster particles. The particles exhibited strong SHG signals, while no fluorescence signals were detected from the gelatin-based film (see second row in Figure 3). Given that SHG signals are specific to non-centrosymmetric structures, we verified that the plaster particles were composed of bassanite crystals using X-ray diffraction analysis. Indeed, among the three types of calcium-sulfate particles (anhydrite, bassanite and gypsum, depending on the water content), only bassanite is a non-centrosymmetric crystal. These data show that SHG microscopy allows the discrimination of the different types of calcium-sulfate crystals as a function of the crystal structure. The ability to specifically image bassanite particles could be precious for conservation studies of works of art containing calcium-sulfate-based materials, for example the preparation layers (gesso) encountered on Italian panel paintings [12].

These results in model monolayer samples show that the two modes of contrast used (2PEF and SHG) allow the discrimination of different materials. Moreover, 3D mapping of the pigments and binder in the various layers was obtained thanks to the optical sectioning of multiphoton microscopy. We were also able to measure the film thickness, to discriminate various fillers and to determine the spatial repartition of the different particles [7].



Model sample

Gelatin-based film containing plaster particles

Sandarac film containingcochineal lake pigments

No SHG signals

No 2PEF signals No 2PEF signals

2PEF ~ 485 nm 2PEF ~ 630 nm SHG

Figure 3. Multiphoton imaging of monolayer model samples. First row: cochineal lake pigments (2PEF signals around 485 nm) in sandarac film (2PEF signals around 630 nm). Second row: plaster particles (SHG signals) in gelatin-based film (no fluorescence signals). Scale bar: 100 µm.

3.3 Model multilayered coating

Stratified layers composed of the previous fillers were studied as model samples for historical coatings. The sample shown in Figure 4 was composed of maple wood coated with a gelatin-based film containing plaster particles as a first layer, and with gelatin-based film containing cochineal lake pigments as a second layer. The stratigraphy of the sample is depicted in Figure 4.d. Multiphoton microscopy provided transverse optical sections at increasing depth. We showed that cochineal lake pigments were revealed by 2PEF signals in the upper layer (Figure 4.a) and plaster particles were observed deeper thanks to SHG signals (Figure 4.b). Finally, we were able to characterize the wood structure through 80 µm thick varnish (Figure 4.c), opening the way to wood characterization through coatings with a thickness of several tens of micrometers. Axial reconstruction was performed and provided the stratigraphy of the sample (Figure 5.e). Finally, a 3D reconstruction was realized to illustrate the potential of multiphoton microscopy for spatial localization of the different fillers (cochineal lake pigments through 2PEF signals in red and plaster particles through SHG signals in green). Moreover, the fine localization of cellulose within the wood cell walls could be characterized.

Another stratified sample with sandarac film was also studied [7]. These results clearly demonstrated the potential of multiphoton microscopy to specifically detect and locate different components in multilayered coating by combining SHG and 2PEF modes of contrast and using various fluorescence detection spectral bands.



Axial reconstruction

3D reconstruction

Transverse imagesz = 40 µm

z = 70 µm

z = 90 µm

L1: cochineal lake in gelatin-based filmL2: plaster in gelatin-based filmL3: wood

L1L2

L3

a

b

c

d

e

f

Figure 4. Multiphoton imaging of multilayered coating on wood. (a-c) Merged 2PEF (in red) and SHG (in green) transverse images from a stratified sample at depths of (a) 40 µm, (b) 70 µm and (c) 90 µm. (d) Scheme of the stratified model sample. (e) Axial reconstruction, showing SHG signals from the cellulose in wood cell walls in the white circle. (f) 3D reconstruction allowing the discrimination and the spatial localization of the particles and the wood fibers. Scale bar: 50 µm.

3.4 In situ investigation of a historical violin

Finally, we demonstrated that multiphoton microscopy can be used for in situ investigation of a historical violin without any observed damage for the instrument. Multiphoton images were compared to conventional optical microscopy and fluorescence microscopy of the same area for a better understanding of the different features [7].

4. CONCLUSION To conclude, this study demonstrates that multimodal multiphoton microscopy is a powerful technique for 3D in situ investigation of historical artifacts and woods. By using several spectral bands, fluorescent materials can be discriminated by the distributions of their fluorescence emission, as shown here for sandarac resin and cochineal lake pigments. SHG signals were detected from plaster. Finally, multiphoton microscopy allows a fine spatial characterization of the wood cell walls through 2PEF signals from lignin and SHG signals from cellulose. Moreover, using excitation wavelength in the near-infrared, this technique allows both imaging of quite deep structures (ca. 90 µm) and reduced possible photo-irradiation damages of the sample compared to conventional (one-photon) fluorescence imaging that



would use excitation wavelengths in the UV-blue region. In practice, no damage was observed in our experimental conditions (laser power below 20 mW at the focal point) in any of the studied model samples and violin.

This study paves the way for numerous promising applications to the fields of ancient materials and conservation science, as well as, more generally, to the fields of coating materials and wood science.

REFERENCES

[1] Targowski, P. and Iwanicka, M., “Optical coherence tomography: its role in the non-invasive structural examination and conservation of cultural heritage objects—a review,” Appl. Phys. A 106, 265–277 (2012).

[2] Latour, G., Echard, J.-P., Soulier, B., Emond, I., Vaiedelich, S., and Elias, M., “Structural and optical properties of wood and wood finishes studied using optical coherence tomography: application to an 18th century Italian violin,” Appl. Opt. 48(33), 6485–6491 (2009).

[3] Latour, G., Moreau, J., Elias, M., and Frigerio, J.-M., “Micro-spectrometry in the visible range with full-field optical coherence tomography for single absorbing layers,” Opt. Comm. 283(23), 4810 – 4815 (2010).

[4] Latour, G., Georges, G., Siozade, L., Deumie, C., and Echard, J.-P., “Study of varnish layers with optical coherence tomography in both visible and infrared domains,” Proc. SPIE 7391(1), 73910J (2009).

[5] Filippidis, G., Massaouti, M., Selimis, A., Gualda, E., Manceau, J.-M., and Tzortzakis, S., “Nonlinear imaging and thz diagnostic tools in the service of cultural heritage,” Appl. Phys. A 106, 257–263 (2012).

[6] Latour, G., Gusachenko, I., Kowalczuk, L., Lamarre, I., and Schanne-Klein, M., “In vivo structural imaging of the cornea by polarization-resolved second harmonic microscopy,” Biomed. Opt. Express 3(1), 1–15 (2012).

[7] Latour, G., Echard, J.-P., Didier, M., and Schanne-Klein, M.-C., “In situ 3d characterization of historical coatings and wood using multimodal nonlinear optical microscopy,” Opt. Express 20(22), 24623–24635 (2012).

[8] Cox, G., Moreno, N., and Feijo, J., “Second-harmonic imaging of plant polysaccharides,” J. Biomed. Opt. 10(2), 024013 (2005).

[9] Gusachenko, I., Tran, V., Goulam-Houssen, Y., Allain, J.-M., and Schanne-Klein, M.-C., “Polarization-resolved second harmonic generation in tendon upon mechanical stretching,” Biophys. J. 102(9), 2220–2229 (2012).

[10] Clementi, C., Doherty, B., Gentili, P., Miliani, C., Romani, A., Brunetti, B. G., and Sgamellotti, A., “Vibrational and electronic properties of painting lakes,” Appl. Phys. A 92(1), 25–33 (2008).

[11] Nevin, A., Echard, J.-P., Thoury, M., Comelli, D., Valentini, G., and Cubeddu, R., “Excitation emission and time-resolved fluorescence spectroscopy of selected varnishes used in historical musical instruments and paintings,” Talanta 80(1), 286–293 (2009).

[12] Martin, E., Sonoda, N., and Duval, A. R., “Contribution à l’étude des préparations blanches des tableaux italiens sur bois,” Stud. Conserv. 37(2), 82–92 (1992).



Documents

SPIE Proceedings [SPIE SPIE Optical Metrology 2013 - Munich, Germany (Monday 13 May 2013)] Optics for Arts, Architecture, and Archaeology IV - Multiphoton microscopy: an efficient